The present invention relates to assays for collagen fragments in body fluids.
Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased susceptibility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15-20 million individuals in the USA alone. Its basis is an age-dependant imbalance in bone remodelling, i.e. in the rates of formation and resorption of bone tissue.
In the USA about 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $10 billion annually in the USA (Riggs, New England Journal of Medicine, 327:620-627 (1992)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited. Other disorders entailing (and correlated with) abnormalities in collagen metabolism include Paget""s disease, Marfan""s syndrome, osteogenesis imperfecta, neoplastic growth in collagenous tissue, dwarfism, rheumatoid arthritis, osteo-arthritis and vasculitis syndrome.
Three known classes of human collagen have been described to date. The Class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils. The amino-acid sequence of type I-III (to the extent it has been elucidated) is given in Appendix A of WO 95/08115.
Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteo-arthritis and collagen type III degradation in vasculitis syndrome.
Amino acid sequences of human type III collagen, human pro a1 (II) collagen, and the entire prepro a1 (III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers; see Loil et al., Nucleic Acid Research 12:9383-9394 (1984): Sangiorgi et al., Nucleic Acids Research, 13:2207-2225 (1985); Baldwin et al., Biochem J., 262:521-528 (1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989).
Type I, II, and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-terminal propeptide sequences, which are attached to the core collagen molecules. After removal of the pro-peptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecules consists largely of a triple-helical domain having terminal telopeptide sequences which are non-triple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extra-celluarly. The alpha-helical region also includes cross-linkable sites.
Intermolecular cross-links provide collagen fibrils with biomechanical stability. The formation of these cross-links is initiated by modification of lysine and hydroxylysine residues to the corresponding aldehydes. Several of these residues located on adjacent chains of collagen will spontaneously form different intermolecular cross-links. The exact position of the sites for cross-linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kxc3xchn, K., in Immunochemistry of the extracellular matrix, 1:1-29, CRC Press, Inc., Boca Raton, Fla. (1982), Eyre, D. R., Ann.Rev. Biochem., 53:717-48 (1984) or U.S. Pat. Nos. 5,140,103 and 5,455,179. Furthermore, the amino acid sequences of some potential sites for cross-linking in type I, II, and III collagen are given in Table 1 below.
The fibrous proteins, collagen and elastin, are cross-linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysine side chains. Four homologous loci of cross-linking are evident in molecules of type I, II and III collagens (for review see Kxc3xchn, K., in Immunochemistry of the extracellular matrix, 1:1-29 (1982)). Two are aldehyde sites, one in each telopeptide region. The other two sites are hydroxylysine symmetrically placed at about 90 residues from each end of the molecule. When collagen molecules pack into fibrils, these latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules. There is now strong evidence that 3-hydroxypyridinium residues are the mature cross-link coming from hydroxylysine-derived aldehydes. The mature cross-linking residues of the other pathway, i.e. from aldehyde formation of lysine residues, are however, still unknown.
As illustrated by formula in EP-0394296 discussed below, the two 3-hydroxypyridinium cross-links have been found to be hydroxylysyl pyridinoline (also known simply as xe2x80x9cpyridinolinexe2x80x9d) and lysyl pyridinoline (also known as xe2x80x9cdeoxypyridinolinexe2x80x9d). These cross-linking compounds are naturally fluorescent. Some hydroxylysyl pyridinoline cross-link are found to be glycosylated as discussed for instance in EP-A-0424428.
However, as described in Last et al, Int. J. Biochem. Vol. 22, No. 6, pp 559-564, 1990 other crosslinks occur naturally in collagen.
In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen.
For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget""s disease, a metabolic bone disorder in which bone turnover is greatly increased, as discussed further below.
For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation; Singer, F. R. et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.), 489-575 (1978), Academic Press, New York.
U.S. Pat. No. 3,600,132 discloses a process for the determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. The Patent states that hydroxyproline correlates with increased collagen anabolism or catabolism associated with patho-logical conditions such as Paget""s disease, Marfan""s syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism.
Bone resorption associated with Paget""s disease has also been monitored by measuring small peptides containing hydroxyproline, which are excreted in the urine following degradation of bone collagen; Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5, 2250262 (1981), and Singer, F. R., et al., supra.
In the case of Paget""s disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index for bone degradation. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline.
Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolised in the liver and never appears in the urine. Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good marker for osteoporosis as it is specific for collagen in bones even if it is not specific for bone resorption, but it is trouble-some to handle.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally non-specific markers of bone resorption; Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).
Other researchers have measured the cross-linking compound 3-hydroxypyridinium in urine as an index of collagen degradation in joint diseases. See, for back-ground and as examples, Wu and Eyre, Biochemistry, 23:1850 (1984): Black et al., Annals of the Rheumatic Diseases, 45:969-973 (1986); and Seibel et al., The Journal of Dermatology, 16:964 (1989). In contrast to the present invention, these prior researchers have hydrolysed peptides from body fluids and then looked for the presence of free 3-hydroxypyridinium residues.
Assays for determination of the degradation of type I, II, and III collagen are disclosed in EP-0394296 and U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103. However, these Patents are restricted to collagen fragments containing the cross-linker 3-hydroxypyridinium. Furthermore, the above mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.
Until recently very few clinical data using the approach described in U.S. Pat. No. 4,973,666 and U.S. Pat. No. 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above mentioned patents) of 3-hydroxypyridinium containing telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densitometry) had been published. Very recently however McClung et al (JBMR(1996)11:129) have concluded that results from the commercial NTx assay based on these Patents do not correlate to bone loss. More particularly, NTx did not correlate to bone loss in the normal population and also failed to predict bone changes in response to therapy. Gertz et al (JBMR (1994) 9(2): 135-142) have reported no significant correlation between baseline NTx measurements and bone loss and no significant correlation between change in NTx and change in bone loss during anti-resorptive therapy. Garnero et al (JBMR (1996) 11(10): 1531-1537) have reported that NTx was found not be predictive of hip fracture whilst other biochemical markers were associated with an approximately 100 percent increased risk of hip fracture.
The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and it would be desirable to avoid this dependence of the correct formation of the cross-linking structure.
GB Patent Application No. 2205643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
Schrxc3x6ter-Kermani et al., Immunol. Invest. 19:475-491 (1990) describe immunological measurement systems based on CNBr fragments of collagen type I and II. Use is made of pepsin-solubilised collagen, leaving the telopeptides in the tissue (cf. the above mentioned GB Patent Application No. 2205643). There is therefore no conformity between the fragments and the antibodies raised therefrom. Further, the reference only describes measurements on extracted tissue samples.
The development of a monoclonal antibody raised against pepsin-solubilised type I collagen is described in Werkmeister et al., Eur. J. Biochem. 1987:439-443 (1990). The antibody is used for immunohistochemical staining of tissue segments and for measuring the collagen content in cell cultures. The measurements are not carried out on body fluids.
EP Patent Application No. 0505210 describes the development of antibody reagents by immunisation with purified cross-linked C-terminal telopeptides from type I collagen. The immunogen is prepared by solubilising human bone collagen with bacterial collagenase. The antibodies thus prepared are able to react with both cross-linked and non-cross-linked telopeptides, and cross-linkers other than pyridinoline.
There are a number of reports indicating that collagen degradation can be measured by quantitating certain procollagen peptides. Propeptides are distinguished from telopeptides and alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see U.S. Pat. No. 4,504,587; U.S. Pat. No. 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al., European Journal of Clinical Investigation, 9:451-459 (1979). EP Patent Application No. 0298210 and No. 0339443 both describe immunological determination of procollagen peptide type III and fragments thereof. Further, a method based on the measurement of procollagen is disclosed in EP Patent Application No. 0465104.
The use of synthetic peptides with sequences derived from type IX collagen for the development of immunological reagents is disclosed in PCT Patent Application No. WO 90/08195. Likewise the application describes the use of the antibodies thus produced for the determination of type IX collagen fragments in body fluids.
U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof in a synovial fluid sample.
Dodge, J. Clin Invest 83:647-661 (1981) discloses methods for analysing type II collagen degradation utilising a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. The degradation products of collagen were not detected in a body fluid, but histochemically by staining of cell cultures, i.e. by xe2x80x9cin situxe2x80x9d detection.
WO 94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments in a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-terminal or N-terminal domain of collagen is incubated with an antibody to the linear synthetic peptide and the sample, and wherein the binding of the antibody to the binding partner is determined.
WO 95/08115 relates to assay methods in which collagen fragments in a body fluid are determined by reaction with an antibody which is reactive with a synthetic peptide. The assay may be a competition assay in which the sample and such a peptide compete for an antibody, possibly a polyclonal antibody raised against fragments of collagen obtained by collagenase degradation of collagen. Alternatively, it may be an assay in which an antibody, possibly a monoclonal anti-body, is used which has been raised against such a synthetic peptide.
As disclosed in WO 91/08478, one particular type of peptide fragment found in body fluid, particularly urine, is of the formula: 
In the above formula, K-K-K is disclosed as representing a hydroxypyridinium cross-link but in fact it may be any naturally occurring cross-link and specifically any of those discussed in the above referenced paper of Last et al.
As further discussed below, larger peptide fragments including the above smaller fragment are also disclosed in this document.
A proportion of the xe2x80x9cpeptidexe2x80x9d fragments in body fluid are related to peptides of equivalent amino acid sequence, e.g. peptides of formula 1, by the isomerisation of aspartic acid in the formula to isoaspartic acid. We put xe2x80x9cpeptidesxe2x80x9d in quotes here as of course the isomerisation means that these species are no longer properly regarded as being peptides.
The isomerisation of proteins containing aspartic acid has been reported previously to be a spontaneous reaction occurring under physiological conditions.
See for instance Brennan et al Protein Science 1993, 2, 331-338, Galletti et al, Biochem. J. 1995, 306, 313-325, Lowenson et al, Blood Cells 1988, 14, 103-117 and Oliya et al, Pharmaceutical Research, Vol. 11, No. 5, 1994, p.751.
The isomerisation has the effect of transferring that part of the peptide chain which runs downstream of the aspartic acid residue in the carboxy terminus direction from the alpha carboxylic acid of the aspartic acid to which it is bonded via a peptide bond in the normal protein to the side chain carboxylic acid in a non-peptide amide bond, as shown below: 
The non-peptide bonded aspartic acid residue is termed xe2x80x9cisoaspartic acidxe2x80x9d or xcex2-aspartic acid (xcex2D).
Similar isomerisation can occur in proteins containing asparagine residues (i.e. with xe2x80x94NH2 instead of xe2x80x94OH in the starting protein in the above reaction scheme).
The above discovery indicates that this isomerisation also occurs in bone tissue and the extent of isomerisation is expected therefore to be marker for the age of the bone tissue concerned.
Furthermore, the presence amongst such bone peptide fragments of the isomerised peptides provides confirmation that the fragments indeed derive from bone degradation and not some other source such as the degradation of newly formed collagen never incorporated into bone.
J. Macek and M. Adam xe2x80x9cDetermination of collagen degradation products in human urinexe2x80x9d, Z. Rheumatol. 46:237-240 (1987) reports the presence of pyridinoline containing collagen cross-linked peptides in urine having a molecular weight above 10,000 but provides no sequence information relating to the peptide chains present or the collagen type to which the fragments belong.
As mentioned above WO 91/08478 discloses that a number of fragments of type 1 collagen can be found in urine. These include a pyridinium crosslink, which may be hydroxylysyl pyridinoline or lysyl pyridinoline. Attached to the crosslink are peptide chains of defined sequence derived from the collagen molecule. The crosslink has three points at which it may bear peptide chains. The fragment of Formula 1 above (Formula VI in WO 91/08478) bears two chains, each having the sequence EKAHDGGR (SEQ ID No.1). Two other fragments are described in WO 91/08748 which each have a third chain, that shown in Formula IV of that specification being 7 amino acids longer than that in Formula V, but otherwise of the same sequence. The amino-acid sequence of the chains of type 1 collagen has been published elsewhere as described above, as has the location of the trivalent crosslinks between the collagen molecules. The third chain depicted in the said Formulae IC and V of WO 91/08748 does not correspond to that of any collagen chain at the location of the crosslink and is believed to be an error, possibly caused by an artefact of the isolation procedure used.
The only fragment for which a credible formula has been given is there for that of Formula VI (equivalent to Formula 1 herein) having two identical peptide chains.
DE-A-4225038 discloses sandwich assays for collagen breakdown products in body fluids. Antibodies are to be produced by immunisation with haptens containing a linear sequence of amino acids. One proposed sequence is FDFSFLP (SEQ ID No.2) and another is PPQEKAHDGGR (SEQ ID No.3), although these were not suggested for use in combination to make two antibodies for use in the same sandwich assay. Indeed although the sequence PPQEKAHDGGR (SEQ ID No.3) is given, no antibodies made against it are specifically described and therefore no disclosure is provided of their actual utility and properties. The only specific sandwich assay described combines an antibody against the C-terminal sequence FDFSFLP (SEQ ID No.2) with one against the sequence GMKGHRGF (SEQ ID No.4) (from the helical region crosslink site).
However, DE-A-4225038 asserts that there is a close correlation between results obtained using an assay based on the sequence FDFSFLP (SEQ ID No.2) and a commercial assay known as the ICTP assay. It has been shown however that the ICTP assay in serum does not appear to reflect bone resorption in that the results it produces do not successfully track the effect of therapeutic treatment (Hassager et al. Calcif. Tissue. Int. (1994) 54:40-33). This of course would imply that the population of molecules containing the sequence FDFSFLP (SEQ ID No.2) in serum would not reflect bone resorption in a useful way.
We have now established that body fluids do in fact contain larger collagen fragments containing not only the sequence EKAHDGGR (SEQ ID No. 1) but also further amino acid residues. These may be present in a third chain attached to the crosslink with two chains incorporating the sequence EKAHDGGR (SEQ ID No. 1) and/or as extensions of N-amino terminal direction of the sequence EKAHDGGR (SEQ ID No. 1) of one or both of the two chains containing that sequence.
We have further established that surprisingly it is possible to obtain binding of two distinct antibodies to a single collagen degradation fragment where both antibodies are specific for an epitope in the sequence EKAHDGGR (SEQ ID No.1) or a variant of it.
Accordingly, the present invention provides a method of measurement of the rate of type I collagen resorption comprising measuring in a sample (e.g. a body fluid) the amount of a population of collagen fragments by a sandwich assay using a first antibody reactive with a first epitope located in the collagen amino acid sequence EKAHDGGR (SEQ ID No.1) or in isomerised and/or racemised variants thereof and a second antibody reactive with a second collagen epitope located in said fragments.
Preferably, the body fluid on which the assay is conducted is other than urine.
Preferably said epitope is treated in the sequence AHDGGR or a said variant of it (SEQ ID No.7).
Optionally, said second epitope is located in the N-amino terminal direction with respect to said first epitope in the same or a different collagen chain. If so, it may include at least a part of the amino acid sequence FDFSF (SEQ ID No. 8).
The assay may therefore be based on the sequence FDFSFLP (SEQ ID No.2) and on the sequence EKAHDGGR (SEQ ID No.1).
Preferably the molecular weight of fragments detected in the assay exceeds 1500 Da, more preferably 5000 Da. However, the molecular weight of the fragments may exceed 10000 Da or even 25000 Da.
Said second collagen epitope is preferably also located in the amino acid sequence EKAHDGGR (SEQ ID No. 1) or in isomerised and/or racemised variants thereof. Each said epitope is therefore preferably present in a respective amino acid chain attached to a crosslink. Preferably each said epitope is located in the amino acid sequence EKAH-xcex2D-GGR (SEQ ID No.5). The antibodies employed are preferably in each case a monoclonal antibody raised against a peptide analogue containing the amino acid sequence EKAH-xcex2D-GGR (SEQ ID No.5).
The invention includes a sandwich assay for collagen degradation products in which antibodies of essentially identical specificity are used on both sides of the sandwich, e.g. antibodies each specific for the same amino acid sequence within the C- or N- telopeptide region of collagen, especially collagen type I or type II or type III.
One or both antibodies may be mAb1H11 described in WO96/36645 or may have similar specificity thereto, i.e. specificity for an epitope contained in the metabolite with which said in mAb1H11 is immunologically reactive.
In a further aspect, the invention includes a method of measurement of the concentration of collagen degradation products in a sample comprising conducting a sandwich assay using first and second immunological binding partners (which may be the same or different) each immunologically reactive with an epitope in an N-terminal telopeptide fragment produced upon collagen degradation in vivo. Such N-terminal fragments may be as described in U.S. Pat. No. 5,455,179 and one or both of the antibodies may be mAb1H11 or have equivalent specificity. The second antibody may be reactive with an eptiope containing amino acid sequence crosslinked in collagen type I degradation products with the mAb1H11 epitope sequence. Accordingly, the fragments may be Type I collagen fragments. Preferably, at least one of the antibodies in specific for an epitope containing an isomerised aspartic acid or asparagine residue.
The preparation of antibodies reactive with the sequence EKAHDGGR has been described in WO95/08115, including methods for the preparation of monoclonal antibodies. As described in WO96/30765 this sequence may be isomerised at the aspartate residue such that this is linked to the following glycine by its xcex2 carboxylic acid group. The preparation of monoclonal antibodies to that isomerised sequence is taught there. As described in PCT/EP97/04372 either the normal sequence or the isomerised sequence may become racemised at the aspartate residue so that one or both chains contains a D-aspartic acid residue either bonded normally or via the xcex2 carboxylic acid group. The production of monoclonal antibodies to the normal or iso-D forms of the sequence may be carried out by processes analogous to those described in WO95/08115 or in WO96/30765. Any of these monoclonal antibodies may be used in the present invention as either or both of the two antibodies, which may have identical specificity. Generally, one antibody will be coupled to a substrate, e.g. a solid or particulate support, and the other will be coupled to a direct or indirect label.
The second antibody for use in the invention may be a monoclonal antibody produced by immunisation with bacterial collagenase treated collagen (CTC) followed by selection for antibodies reactive with a selected peptide sequence such as that set out below taken from the sequence of the collagen molecule upstream (i.e. in the N-terminal direction) from the sequence EKAHDGGR (SEQ ID No.1). Preferably in this case, the sequence is one which embraces the immunogenic sequence FDFSFL (SEQ ID No.2). A preferred peptide for this purpose is one that contains the sequence FDFSFL (SEQ ID No.2) plus additional amino acids at one or more preferably both ends such as CSAGFDFSFLPQPPQE (SEQ ID No.6).
Alternatively, immunisation may be carried out with the peptide itself conjugated to a suitable carrier according to known techniques.
Where both epitopes are contained in the same amino acid sequence, e.g. EKAHDGGR (SEQ ID No. 1) or its variants, both antibodies may be the same or may be raised in the same way.
The methods of preparation of monoclonal antibodies are well known in the art. For example, see Campbell, A. M., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 12 (1986). It is possible to produce antibodies to synthetic peptides or their isomerised or racemised variants by immunisation. However, because of the relatively small molecular weight of these compounds it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin, thyroglobulin, ovalbumin, tetanus toxoid, and keyhole limpet haemocyanin. The preferred carrier is bovine serum albumin. To present the hapten in its most immunogenic form to the antibody producing cells of the immunised animal a number of alternative coupling protocols can be used. Suitable procedures include, but are not limited to, glutaraldehyde, carbodiimide, and periodate. Preferred binding agents are glutaraldehyde and carbodiimide.
The preparation of antibodies may also be carried out by conventional techniques including immunisation with collagen fragments which may contain natural isomerisation or with an adjuvant before injection. Examples of adjuvants include, but are not limited to, aluminium hydroxide, Freund""s adjuvant, and immune-stimulating complexes (ISCOMs). ISCOMs can be made according to the method described by Morein, B. et al., Nature 308:457-460 (1984).
Either monoclonal or polyclonal antibodies to the haptencarrier molecule can be produced. For the production of monoclonal antibodies it is preferred that mice are immunised. Spleen cells from the immunised mouse are harvested, homogenised, and thereafter fused with cancer cells in the presence of polyethylene glycol to produce a cell hybrid which produces monoclonal antibodies specific for isomerised peptide fragments derived from collagen. Suitable cancer cells include, but are not limited to, myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of the production of monoclonal antibodies are provided in Goding, J. W., in Monoclonal Antibodies: Principles and Practice, (1986). A preferred preliminary screening protocol comprises the use of synthetic peptides or isomerised or racemised peptide analogues conjugated to a carrier and coated on to the solid surface of a microtitre plate.
For the preparation of polyclonal antibodies, different animal species can be immunised. Suitable species include, but are not limited to, chicken, rabbit and goat. Chicken and rabbit are preferred.
Antibodies so produced may be screened for suitability for use according to the invention by testing for reactivity with a synthetic peptide or peptide analogue of appropriate sequence.
Antibody fragments are prepared by methods known in the art (see E. Ishikawa. Journal of Immunoassay 3:209-327 (1983)).
It is possible to omit (or add) one or more amino acid residues from (or to) the crosslink site sequences without substantial loss of the ability to raise antibodies recognising the corresponding native collagen fragment. It is possible to use longer collagen fragments and/or chimeric peptide analogues to raise the antibodies. It is possible to make substitution of one or more amino acids which are not critical for antibody recognition.
The preparation of synthetic peptides and peptide analogues may be performed according to procedures well known in the art, e.g. by solid-phase peptide synthesis techniques commonly described as xe2x80x9cMerrifield synthesisxe2x80x9d. Also classical solution phase techniques may be used.
The sandwich assay according to the present invention may be conducted according to any of the known sandwich assay formats. These include formats in which one antibody is provided on a solid support and the other is labelled in one of many known ways including the use of radio-isotope labels. The antibodies and revealing reagents for the conduct of an immunoassay using standard detection protocols, for example radioisotope labelling, fluorescent labelling or ELISA, may conveniently be supplied as kits which include the necessary components and instructions for the assay. In one embodiment of the invention such a kit includes a microtitre plate coated with a relevant antibody, standard solutions for preparation of standard curve, a body fluid (e.g. serum) control for quality testing of the analytical run, a second antibody reactive with a second epitope in fragments to be detected conjugated to an enzyme such as horse radish peroxidase or otherwise labelled, a substrate solution, a stopping solution, a washing buffer and an instruction manual.
Both antibodies may be bound to a micro-particle, e.g. in a latex emulsion, so that binding of the antibodies to the target produces an agglutination which can be observed by known techniques such as light transmission or scattering measurements.
However, the two antibodies can be mixed with the sample before one of the antibodies is bound to a capture substrate such as a microtitre plate or other form of solid support such as micro-beads. For such use one antibody may be coupled to a capture moiety which has affinity for and can be captured on a capture substrate. It is preferred that the other antibody is coupled to a direct or indirect label.
The capture moiety may for instance be biotin. Biotinylated antibodies may be captured on a capture substrate bearing avidin or streptavidin.
The use of such a format is particularly suitable where each antibody has the same or an overlapping specificity, there being two identical or mutually interfering epitopes in each of the collagen degradation fragments to be detected. Addition of the target fragments to either one of the antibodies separately may result in both epitopes being bound by the first antibody, so that the second antibody when added does not bind. This may occur even when the first antibody is immobilised on a solid surface. If both antibodies are mixed with the target fragments at the same time, sandwiches containing the first antibody, the target fragment and the second antibody can be formed. These may then be captured to a capture substrate by the capture moiety present on one antibody and the captured sandwich can then be detected via the label of the other antibody.
Accordingly the invention provides in a separate aspect a method of conducting a sandwich assay comprising:
mixing a target antigen containing at least two antigenically similar epitopes with a first antibody reactive with both said epitopes, which first antibody is coupled to a capture moiety and with a second antibody reactive with both said epitopes, which second antibody is coupled to a label, so as to form a first antibodyxe2x80x94target antigenxe2x80x94second antibody sandwich, capturing said sandwich to a capture substrate having an affinity for said capture moiety of said first antigen,
and detecting the capture of said sandwich by detection of the label of the second antibody.
Since immunoassays can be constructed using antibodies specific for synthetic isomerised and/or racemised peptide analogues, the ratios of the corresponding collagen fragment sequences in an appropriate biological fluid can be determined as well as their individual levels and their total. Thus, the assay can be designed to include antibodies which will result in determination of fragments containing several isoaspartic acid containing and/or racemised peptide analogues and optionally the native peptide sequences or determination of a single isoaspartic acid containing and/or racemised peptide analogue sequence, or any desired combination thereof.
In addition to the use of the herein specified peptides as indicators of bone resorption, bone metabolic balance is advantageously determined by the substantially simultaneous determination of a marker of the formation of bone in the same or other appropriate biological fluid form the same individual. xe2x80x9cSubstantially simultaneousxe2x80x9d means the same day, preferably within 4 hours. For example such markers include osteocalcin (also known as bone GLA protein of BGP), propeptides of procollagen type I, bone alkaline phosphatase and total alkaline phosphatase. Suitable methods for the determination of these markers can be found, for example, in Delmas, P. D., et al., J. Bone Min. Res. (1986) 1:333-337.
The assay of the present invention which provides an index to determination of the metabolic status of tissues, which generate collagen-derived peptides and isomerised and/or racemised peptide analogues when degradation occurs, is useful in a variety of contexts. The assays may be used to assess an abnormal condition of a subject by indicating excessive bone resorption. This may show the presence of an osteoporotic condition or the metastatic progress of an malignancy. Other conditions characterised by excessive bone resorption include Paget""s disease and hyperparathyroidism.